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Steric-Free Bioorthogonal Labeling of Post-translational Modification Substrates Based on A Fluorine-Thiol/Selenol Displacement ReactionLyu, Zhigang, 0000-0002-6903-3965 January 2021 (has links)
Post-translational modifications (PTMs) diversify the functions and control the stability of proteins by the covalent addition of chemical groups or proteins. These modifications include phosphorylation, acetylation, glycosylation, methylation, ubiquitination, etc. and affect all aspects of cellular activities. Dysregulation of PTMs is often linked to aging, oncogenesis, and various autoimmune diseases. Proteins involved in the PTM writing, removing, and reading process are very important biomarkers and hot therapeutic targets. Yet, current chemical methodologies to globally profiling PTM substrates rely on coppercatalyzed azide-alkyne cycloaddition (CuAAC) reaction in which bulky alkyne or azide groups in length and size are used. The intrinsic steric hinderance limits the general application of chemical reporters to metabolic incorporation by PTM writers possessing spacious active pockets.This dissertation will start with the demonstration that the alpha fluorine to amide bond was a steric free chemical reporter in protein acetylation. Fluorinated cofactors or precursors could be utilized by acetyltransferases to modify peptides and proteins both in vitro and in live cells. A novel bioorthogonal reaction named as fluorine thiol displacement reaction
(FTDR) using thiophenol as the warhead was developed for converting a fluorine label in biomolecules to a fluorophore or biotin tag. This whole platform showed great potential to label, image, and enrich acetylation substrates.
Another goal of this dissertation was the evolution of FTDR to fluorine selenol displacement reaction (FSeDR). Aliphatic selenol, aromatic selenol and thiol derivatives were compared in parallel in the labelling of fluorinated small molecules and proteins. Aliphatic selenol displayed the best reactivity under the mild physiological condition and exhibited a high degree of chemical stability. The updated platform was then used in the SILAC-based quantitative proteomics study of acetylation in the prostate cancer cell PC-3.
The final goal of this dissertation focuses on the study of steric-free bioorthogonal labeling of glycosylation substrates based on FSeDR. The fluorine-tag labelled unnatural monosaccharides could be utilized by glycotransferases in live cells to modify sialylated glycoproteins, N-glycoproteins, and O-GlcNAcylated proteins. With the help of FSeDR, the fluorine reporter was further functionalized to fluorescent probes or affinity tags for imaging or enrichment studies. The steric-free fluorine reporter has the potential to become a powerful chemical reporter for glycan labelling. / Chemistry
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The role of PPARgamma acetylation and Adipsin in adipose tissue dysfunctionAaron, Nicole January 2022 (has links)
Adipose tissue is a key metabolic organ responsible for maintaining energy homeostasis throughout the body. Healthy adipocytes respond to physiological changes and perform a variety of important functions to regulate glucose and lipid metabolism. Dysregulation of adipose tissue function, on the other hand, is strongly associated with the development of metabolic diseases. Peroxisome Proliferator Activated Receptor gamma (PPARγ) is a key transcription factor that regulates various activities in adipocytes as well as other cell types. A growing body of evidence indicates a more complex role for PPARγ beyond its classical ligand-dependent activity, including the exploration of posttranslational modifications and associated target proteins in non-canonical adipogenic reservoirs and adipocyte-associated cells.
The first part of the thesis describes our study identifying Adipsin as a downstream target of PPARγ deacetylation and further uncovers its function within the bone marrow niche. Unlike peripheral adipose tissues, marrow adipose tissue has been shown to be uniquely responsive to nutrient fluctuations, hormonal changes, and metabolic disturbances such as obesity and diabetes mellitus. Expansion of marrow adipose tissue has also been strongly associated with bone loss in mice and humans. However, the regulation of bone marrow plasticity remains poorly understood, as does the mechanism that links changes in marrow adiposity with bone remodeling. We show that Adipsin was robustly induced in the bone marrow during bone loss in mouse and humans, in a manner dependent on PPARγ acetylation. Ablation of Adipsin inhibited marrow adipose expansion and improved skeletal health in bone loss conditions of calorie restriction, thiazolidinedione treatment for insulin resistance, and aging. These effects were mediated by Adipsin’s downstream effector, Complement Component 3, to prime common progenitor cells toward adipogenesis rather than osteoblastogenesis through the inhibition of Wnt/β-catenin signaling. Together, our findings reveal an unknown function of Adipsin, mediated by PPARγ acetylation, to promote adiposity and affect skeletal remodeling in the bone marrow niche.
The second part of the thesis addresses another novel role for PPARγ, through acetylation in macrophages, to promote adipose tissue inflammation. Chronic, low-grade inflammation characteristic of obesity and metabolic dysfunction is partially driven by macrophage infiltration of adipose tissue and associated inflammatory signaling. PPARγ plays a critical role in regulating anti-inflammatory, M2 polarization of macrophages. However, the involvement of post-translational modifications, such as acetylation, in macrophages is unknown. Here we generated a macrophage specific, PPARγ constitutive acetylation-mimetic mouse line (K293Qflox/flox;LysMcre, mK293Q) to dissect its role. Upon stimulating macrophage infiltration into adipose tissue by high-fat diet feeding, we assessed the overall metabolic profile and tissue-specific phenotype of the mutant mice. We found that the mK293Q mutant promotes pro-inflammatory macrophage infiltration and subsequent fibrosis specifically in epididymal but not subcutaneous white adipose tissue, driving an impaired metabolic response including decreased energy expenditure, insulin sensitivity, glucose tolerance, and adipose tissue function. These detriments are driven by suppressed anti-inflammatory activation of macrophages. Furthermore, mK293Q mice are resistant to improvements in adipose remodeling by Rosiglitazone treatment. Our study reveals acetylation as a new layer of PPARγ regulation in macrophage activation. These findings highlight the importance of post-translational modifications in determining the function of PPARγ when regulating metabolism and promote the discovery of anti-inflammatory associated therapeutics.
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Role of Nuclear Hat1p Complex and Acetylation of Newly Synthesized Histone H4 in Chromatin AssemblyGe, Zhongqi 20 May 2013 (has links)
No description available.
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Histone Deacetylase 6 (HDAC6) Is Critical for Tumor Cell Survival and Promotes the Pro-Survival Activity of 14-3-3ζ viaDeacetylation of Lysines Within the14-3-3ζ Binding PocketMortenson, Jeffrey Benjamin 01 July 2015 (has links) (PDF)
Our understanding of non-histone acetylation as a means of cellular regulation is in its infancy. Using a mass spectrometry approach we identified acetylated lysine residues and monitored acetylation changes across the proteome as a consequence of metabolic stress (hypoxia). We observed changes in acetylation status of non-histone lysines in tumor cells. Through the use of small molecule inhibitors of histone deacetylase enzymes (HDACs) and siRNA screening identified HDAC6 as a pro-survival regulator of lysine acetylation during hypoxia. The phospho-binding protein 14-3-3ζ acts as a signaling hub controlling a network of interacting partners and oncogenic pathways. We show here that lysines within the 14-3-3ζ binding pocket and protein-protein interface can be modified by acetylation. The positive charge on two of these lysines, K49 and K120, is critical for coordinating 14-3-3ζ-phosphoprotein interactions. Through screening, we identified HDAC6 as the K49/K120 deacetylase. Inhibition of HDAC6 blocks 14-3-3ζ interactions with two well-described interacting partners, Bad and AS160, which triggers their dephosphorylation at S112 and T642, respectively. Expression of an acetylation-refractory K49R/K120R mutant of 14-3-3ζ rescues both the HDAC6 inhibitor-induced loss of interaction and S112/T642 phosphorylation. Furthermore, expression of the K49R/K120R mutant of 14-3-3ζ inhibits the cytotoxicity of HDAC6 inhibition. These data demonstrate a novel role for HDAC6 in controlling 14-3-3ζ binding activity.
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A MOLECULAR ‘SWITCHBOARD’-LYSINE MODIFICATIONS AND THEIR IMPACT ON TRANSCRIPTIONZheng, Gang January 2006 (has links)
No description available.
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Inherent Alteration of Histine Acetylation in Cell Culture Models of Cystic FibrosisBartling, Toni Renee 06 October 2008 (has links)
No description available.
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Novel Small Molecules Regulating The Histone Marking, AR Signaling, And AKT Inhibition In Prostate CancerHuang, Po-Hsien 23 August 2010 (has links)
No description available.
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Epigenetic Mechanisms in Blast-Induced NeurotraumaBailey, Zachary S. 06 September 2017 (has links)
Blast-induced neurotrauma (BINT) is a prevalent brain injury within both military and civilian populations due to current engagement in overseas conflict and ongoing terrorist events worldwide. In the early 2000s, 78% of injuries were attributable to an explosive mechanism during overseas conflicts, which has led to increased incidences of BINT [1a]. Clinical manifestations of BINT include long-term psychological impairments, which are driven by the underlying cellular and molecular sequelae of the injury. Development of effective treatment strategies is limited by the lack of understanding on the cellular and molecular level [2a]. The overall hypothesis of this work is that epigenetic regulatory mechanisms contribute to the progression of the BINT pathology and neurological impairments. Epigenetic mechanisms, including DNA methylation and histone acetylation, are important processes by which cells coordinate neurological and cellular response to environmental stimuli. To date, the role of epigenetics in BINT remains largely unknown.
To test this hypothesis, an established rodent model of BINT was employed [3a]. Analysis of DNA methylation, which is involved in memory processes, showed decreased levels one week following injury, which was accompanied by decreased expression of the enzyme responsible for facilitating the addition of methyl groups to DNA. The one week time point also showed dramatic decreases in histone acetylation which correlated to decline in memory. This change was observed in astrocytes and may provide a mechanistic understanding for a hallmark characteristic of the injury. Treatment with a specific enzyme inhibitor was able to mitigate some of the histone acetylation changes. This corresponded with reduced astrocyte activation and an altered behavioral phenotype, which was characterized by high response to novelty. The diagnostic efficacy of epigenetic changes following blast was elucidated by the accumulation of cell-free nucleic acids in cerebrospinal fluid one month after injury. Concentrations of these molecules shows promise in discriminating between injured and non-injured individuals.
To date, the diagnostic and therapeutic efforts of BINT have been limited by the lack of a mechanistic understanding of the injury. This work provides novel diagnostic and therapeutic targets. The clinical potential impact on diagnosis and therapeutic intervention has been demonstrated. / Ph. D.
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Skeletal Muscle Acetylation in Response to an Acute and Chronic High-Fat DietKavanaugh, John Wesley 11 December 2017 (has links)
The past thirty years have seen a dramatic rise in obesity worldwide owing to a change in dietary composition, quantity of food consumed- positive energy balance, and a more sedentary life style. Accompanied with obesity is a chronic low grade inflammatory state defined by increased circulating cytokines and an increase in gene expression promoting inflammation. Multiple health risks are associated with obesity such as cardiovascular disease, insulin resistance, and type II diabetes.
Advances in mass spectrometry have made wide scale proteomic studies possible and are redefining cell and molecular biology. One such area of that has become of considerable interest is protein acetylation which is observed in most cellular processes such as cell cycle regulation, gene expression, subcellular localization, metabolism, muscle contraction, protein stability, apoptosis, and more. Metabolic proteins are highly susceptible to acetylation with almost all showing the capacity to be acetylated.
Our research, using an obese mouse model fed a chronic high fat diet and a lean control mouse model fed a standard chow diet, showed numerous differences in the acetylome between obese and lean animals in a fasted state. As well as, differences in the acetylome's of both animal models upon receiving a high fat meal. We showed that almost every mitochondrially located metabolic protein in obese animals is hyper-acetylated in a fasted state compared to lean animals and that upon feeding lean animals have a greater response in the change to their metabolic acetylome. We show that in the fed state lean and obese mice have almost completely different acetylomic profiles of mitochondrial and glycolytic metabolic proteins. Furthermore, we have observed possible new regulatory mechanisms utilizing acetylation to 1) determine the fate of the co-factor NADH in glycolysis and 2) control an ATP producing reaction in glycolysis. / Ph. D.
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Lyocell Fiber-Reinforced Cellulose Ester Composites - Surface and Consolidation Considerations, and PropertiesSeavey, Kevin Christopher 09 November 1999 (has links)
The objective of this thesis was to further develop the polymer composite system consisting of cellulose acetate butyrate (CAB) and high modulus, continuous, regenerated cellulose fiber (lyocell). Of particular concern were both the interfacial adhesion between the fiber and matrix and the consolidation process in the manufacture of these composite materials.
Interfacial adhesion was found to be substantial due to the relative lack of the fiber pull-out phenomenon observed after tensile failure in the unmodified fiber composites. This result was then supported in the second study in which similar unmodified fiber composites experienced very little fiber pull out with evidence of a large amount of cohesive failure of the matrix accompanied by matrix particles adhering to the fiber surfaces.
Void volume formation was mitigated to a small extent by the use of optimal consolidation conditions. Composites formed at moderate temperature (200 °C), low consolidation pressure (11.8 p.s.i.) and high consolidation time (13 min.) were found to have the lowest void volume formation of ca. 2.8 %. These composites were generally found to have the highest interfacial shear strength, ca. 16 MPa. A tensile modulus of 22 GPa and an ultimate strength of 246 MPa was obtained for this composite having a fiber volume content of ca. 62 %. / Master of Science
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